1. Field of the Invention
The present invention relates to methods for detecting protein kinase activity and kits for performing such methods.
2. Description of Related Art
Protein kinases play crucial roles in the modulation of a wide variety of cellular events. These enzymes act by transferring phosphate residues to certain amino acids in intracellular polypeptides, to bring about the activation of these protein substrates, and set in motion a cascade of activation controlling events including the growth, differentiation and division of cells. Protein kinases have been extensively studied in the field of tumour biology. A lack of controlled activity of kinases in cells is believed to lead to the formation of tumours. The pharmaceutical industry is constantly in search of drugs that target these kinases, to help with the treatment of a wide variety of tumours. There are at least 1200 protein kinases that are involved in the regulation of cell functions. They occur as both transmembrane and cytosolic enzymes and they phosphorylate serine, threonine and tyrosine amino acid residues. Based on these substrate specificities the kinases are divided into two groups, the serine/threonine kinases and tyrosine kinases. This has led to the development of a number of techniques that focus on the ability of these proteins to take a phosphate group and attach it to a protein/peptide.
One of the most widely used techniques is a radio-isotope method, that utilises either 32P or 33P gamma phosphates. In the presence of an active kinase, the labelled phosphate is transferred from the ATP to the protein or peptide substrate. These assays need to be performed in the presence of ATP labelled to a high specific activity. This results from keeping the concentration of unlabelled ATP in the micromolar concentration range. Also in order to achieve the required sensitivity the peptide substrate has to be used at high concentrations (5-20 μM). The increased radioactivity on the resulting phosphoproteins can then be detected using scintillation counters after capture on phosphocellulose paper.
Other methods include immunoprecipitation procedures. During these assays the kinase, ATP and substrate reaction is allowed to proceed and is then stopped using a buffer, such as Laemmli buffer. The protein is then run out using SDS/PAGE electrophoresis. The gel is then blotted onto a nitrocellulose membrane and probed for phosphorylated substrate, using an antibody to the phosphorylated amino acid of choice. The presence of the phosphorylated band can be visualised using a secondary antibody conjugated to horseradish peroxidase, followed by the use of a chemiluminescence detection system, and exposure onto photographic film. As in the case of many of the methods that have been proposed as an alternative to the radioisotope assays, however, the above western blotting technique lacks sensitivity and is quite laborious.
The use of luminescent detection, either by bioluminescence or chemiluminescence allows for a highly sensitive detection system. Lehel et al. (1997) Anal. Biochem. 244, 340-346 reported the use of a chemiluminescent microtitre plate assay for detection of protein kinase activity. This assay is based on the use of biotinylated substrate peptides captured on a streptavidin-coated microplate, together with monoclonal antibodies. The authors chose protein kinase A (PKA) to develop the assay, but also found reliable results with chose protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II, receptor interacting protein and src activities. These assays were performed in the presence of 20 μM ATP and the kinase of interest +/− inhibitor, the kinase reaction was allowed to proceed to completion. The plates were then washed prior to antibody binding and chemiluminescence detection with a secondary antibody conjugated to horseradish peroxidase with chemiluminescence determined using a Tropix (RTM) (USA) chemiluminescent substrate kit. This assay still relies upon the availability of specific substrates, and also antibodies to the phosphoproteins generated.
Another approach that has been taken, is to adopt microchip-based technology. Cohen et al. (1999) Anal. Biochem. 273, 89-97, reported an assay for PKA based on photolithographic techniques. Performing an on-chip electrophoretic separation of the fluorescently labelled peptide substrate and product allowed for determination of the movement of the phosphate group from ATP to the serine residue of the heptapeptide, Kemptide. This technology was developed for the detection of PKA activity.
Eu et al (1999) Anal. Biochem. 271, 168-176 describe a method in which the measurement of ATP via bioluminescence is related to the amount of substrate (galactose) which is present in a urine sample.
Sala-Newby & Campbell (1992) FEBS Lett 307, 241-244 describe the use of a firefly luciferase which was engineered to contain a protein kinase A recognition site RRFS and to lack the C-terminal peroxisomal signal of native luciferase. The mutant luciferase was expressed in COS cells and used to detect and quantify protein kinase A activation by cyclic AMP in those cells.
It will be appreciated that the above method is extremely specific, being useful only for protein kinase A activation by cyclic AMP. Hence, it suffers from the same problems as the protein kinase detection systems described above which are based on the specific enzymes and substrates with which they react.
There are no assays that have the ability to determine kinase activity irrespective of the kinase family or the amino acid residues that are phosphorylated. This is due to the fact that all the methods currently available focus on the specific enzymes and substrates involved.
The present invention seeks to provide methods for measuring protein kinase activity which are not specific for a single protein kinase, but rather can be used as a general means of measuring the activity of a wide range of protein kinases.